OBJECTIVE

To perform a genome-wide association study (GWAS) in Koreans in order to identify susceptibility loci for rheumatoid arthritis (RA).

METHODS

We generated high-quality genotypes for 441,398 single-nucleotide polymorphisms (SNPs) in 801 RA cases and 757 controls. We then tested 79 markers from 46 loci for replication in an independent sample of 718 RA cases and 719 controls.

RESULTS

Genome-wide significance (P < 5 × 10(-08) ) was attained by markers from the major histocompatibility complex region and from the PADI4 gene. The replication data showed nominal association signals (P < 5 × 10(-02) ) for markers from 11 of the 46 replicated loci, greatly exceeding random expectation. Genes that were most significant in the replication stage and in the combined analysis include the known European RA loci BLK, AFF3, and CCL21. Thus, in addition to the previously associated STAT4 alleles, variants at these three loci may contribute to RA not only among Europeans, but also among Asians. In addition, we observed replication signals near the genes PTPN2, FLI1, ARHGEF3, LCP2, GPR137B, TRHDE, and CGA1. Based on the excess of small P values in the replication stage study, we estimate that more than half of these loci are genuine RA susceptibility genes. Finally, we systematically analyzed the presence of association signals in Koreans at established European RA loci, which showed a significant enrichment of European RA loci among the Korean RA loci.

CONCLUSIONS

Genetic risk for RA involves both population-specific loci as well as many shared genetic susceptibility loci in comparisons of Asian and European populations.

Antibody responses represent a key immune protection mechanism. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset that provides help to B cells to generate an antibody response. Tfh cells together with B cells form germinal centers (GCs), the site where high-affinity B cells are selected and differentiate into either memory B cells or long-lived plasma cells. We show here that interleukin-12 receptor β1 (IL-12Rβ1)-mediated signaling is important for in vivo Tfh response in humans. Although not prone to B cell-deficient-associated infections, subjects lacking functional IL-12Rβ1, a receptor for IL-12 and IL-23, displayed substantially less circulating memory Tfh and memory B cells than control subjects. GC formation in lymph nodes was also impaired in IL-12Rβ1-deficient subjects. Consistently, the avidity of tetanus toxoid-specific serum antibodies was substantially lower in these subjects than in age-matched controls. Tfh cells in tonsils from control individuals displayed the active form of signal transducer and activator of transcription 4 (STAT4), demonstrating that IL-12 is also acting on Tfh cells in GCs. Thus, our study shows that the IL-12-STAT4 axis is associated with the development and the functions of Tfh cells in vivo in humans.

The Jak-STAT pathway was originally discovered through the study of interferon induced intracellular signal transduction. Meanwhile, a large number of cytokines, hormones and growth factors have been found to activate Jaks and STATs. Jaks (Janus Kinases) are a unique class of tyrosine kinases that associate with cytokine receptors. Upon ligand binding, they activate members of the Signal Transducers and Activators of Transcription (STAT) family through phosphorylation on a single tyrosine. Activated STATs form dimers, translocate to the nucleus, bind to specific response elements in promotors of target genes, and transcriptionally activate these genes. Both positive and negative regulations of the Jak-STAT pathway have been identified. In a positive feedback loop, interferons transcriptionally activate the genes for components of the interferon stimulated gene factor 3 (ISGF3). A number of cytokines that activate the Jak-STAT pathway, e.g. IL-6, IL-4, LIF, G-CSF, have been shown to upregulate the expression of SOCS-JABs-SSIs, a recently discovered class of STAT inhibitors. Targeted disruption of genes for a number of Jaks and STATs in mice have revealed specific biological functions for many of them. Although most of the STATs are activated in cell culture by many different ligands, STAT knockout mice mostly show defects in a single or a few cytokine dependent processes. STAT1 knockout mice have an impaired interferon signalling, STAT4 knockouts impaired IL-12 signalling, STAT5a knockouts impaired prolactin signalling, STAT5b knockouts impaired growth hormone signalling, and STAT6 knockout impaired IL-4 and IL-13 signalling. Defects in the Jak-STAT pathway have already been identified in a number of human diseases. Prominent amongst them are leukaemias, lymphomas and inherited immunodeficiency syndromes. It can be expected that additional Jak-STAT related diseases will be identified over the next years. To date, specific STAT inhibitory drugs are not known, but a number of specific protein-protein interactions in the Jak-STAT pathway are potential targets for pharmaceutical interventions.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.

Primary Sjögren's syndrome is one of the most common autoimmune diseases. So far, genetic studies of Sjögren's syndrome have relied mostly on candidate gene approaches. To identify new genetic susceptibility loci for primary Sjögren's syndrome, we performed a three-stage genome-wide association study in Han Chinese. In the discovery stage, we analyzed 556,134 autosomal SNPs in 542 cases and 1,050 controls. We then validated promising associations in 2 replication stages comprising 1,303 cases and 2,727 controls. The combined analysis identified GTF2I at 7q11.23 (rs117026326: Pcombined = 1.31 × 10(-53), combined odds ratio (ORcombined) = 2.20) as a new susceptibility locus for primary Sjögren's syndrome. Our analysis also confirmed previously reported associations in Europeans in the regions of STAT4, TNFAIP3 and the major histocompatibility complex (MHC). Fine mapping of the region around GTF2I showed that rs117026326 in GTF2I had the most significant association, with associated SNPs extending from GTF2I to GTF2IRD1-GTF2I.

A recent study in the North American White population has documented the association of a common STAT4 haplotype (tagged by rs7574865) with risk for rheumatoid arthritis (RA) and systemic lupus erythematosus. To replicate this finding in the Korean population, we performed a case-control association study. We genotyped 67 single nucleotide polymorphisms (SNPs) within the STAT1 and STAT4 regions in 1123 Korean patients with RA and 1008 ethnicity-matched controls. The most significant four risk SNPs (rs11889341, rs7574865, rs8179673, and rs10181656 located within the third intron of STAT4) among 67 SNPs are identical with those in the North American study. All four SNPs have modest risk for RA susceptibility (odds ratio 1.21-1.27). A common haplotype defined by these markers (TTCG) carries significant risk for RA in Koreans [34 percent versus 28 percent, P=0.0027, OR (95 percent CI)=1.33 (1.10-1.60)]. By logistic regression analysis, this haplotype is an independent risk factor in addition to the classical shared epitope alleles at the HLA-DRB1 locus. There were no significant associations with age of disease onset, radiographic progression, or serologic status using either allelic or haplotypic analysis. Unlike several other risk genes for RA such as PTPN22, PADI4, and FCRL3, a haplotype of the STAT4 gene shows consistent association with RA susceptibility across Whites and Asians, suggesting that this risk haplotype predates the divergence of the major racial groups.

Although required for many fundamental immune processes, ranging from self-tolerance to pathogen immunity, interleukin (IL)-2 production is transient, and the mechanisms underlying this brevity remain unclear. These studies reveal that helper T cell IL-2 production is limited by a classic negative feedback loop that functions autonomously or in collaboration with other common gamma chain (IL-4 and IL-7) and IL-6/IL-12 family cytokines (IL-12 and IL-27). Consistent with this model for cytokine-dependent regulation, they also demonstrate that the inhibitory effect can be mediated by several signal transducer and activator of transcription (STAT) family transcription factors, namely STAT5, STAT4, and STAT6. Collectively, these findings establish that IL-2 production is limited by a network of autocrine and paracrine signals that are readily available during acute inflammatory responses and, thus, provide a cellular and molecular basis for its transient pattern of expression.

IL-12 and IL-18 synergistically enhance IFN-gamma mRNA transcription by activating STAT4 and AP-1, respectively. However, it is still unknown how STAT4/AP-1 elicit IFN-gamma promoter activation. Using an IL-12/IL-18-responsive T cell clone, we investigated the mechanisms underlying synergistic enhancement of IFN-gamma mRNA expression induced by these two cytokines. Synergy was observed in a reporter gene assay using an IFN-gamma promoter fragment that binds AP-1, but not STAT4. An increase in c-Jun, a component of AP-1, in the nuclear compartment was elicited by stimulation with either IL-12 or IL-18, but accumulation of serine-phosphorylated c-Jun was induced only by IL-18 capable of activating c-Jun N-terminal kinase. The binding of AP-1 to the relevant promoter sequence depended on the presence of STAT4. STAT4 bound with c-Jun, and a phosphorylated c-Jun-STAT4 complex most efficiently interacted with the AP-1-relevant promoter sequence. Enhanced cobinding of STAT4 and c-Jun to the AP-1 sequence was also observed when activated lymph node T cells were exposed to IL-12 plus IL-18. These results show that STAT4 up-regulates AP-1-mediated IFN-gamma promoter activation without directly binding to the promoter sequence, providing a mechanistic explanation for IL-12/IL-18-induced synergistic enhancement of IFN-gamma gene expression.

The production of IFN-gamma by T cells and the ability of this cytokine to activate the transcription factor STAT1 are implicated in the activation of antimicrobial mechanisms required for resistance to intracellular pathogens. In addition, recent studies have suggested that the ability of STAT1 to inhibit the activation of STAT4 prevents the development of Th1 responses. However, other studies suggest that STAT1 is required to enhance the expression of T-bet, a transcription factor that promotes Th1 responses. To address the role of STAT1 in resistance to T. gondii, Stat1-/- mice were infected with this pathogen, and their response to infection was assessed. Although Stat1-/- mice produced normal serum levels of IL-12 and IFN-gamma, these mice were unable to control parasite replication and rapidly succumbed to this infection. Susceptibility to toxoplasmosis was associated with an inability to up-regulate MHC expression on macrophages, defects in NO production, and the inability to up-regulate some of the IFN-inducible GTPase family of proteins, molecules associated with antitoxoplasma activity. Analysis of T cell responses revealed that STAT1 was not required for the development of a Th1 response, but was required for the infection-induced up-regulation of T-bet. Together these studies suggest that during toxoplasmosis the major role of STAT1 is not in the development of protective T cell responses, but, rather, STAT1 is important in the development of antimicrobial effector mechanisms.

Multiple sclerosis is a chronic inflammatory disease of the CNS. Although progressive axonal injury and diffuse inflammatory damage has been shown in the chronic phase of the disease, little is known about the molecular mechanisms underlying these pathological processes. In order to identify these mechanisms, we have studied the gene expression profile in non-lesion containing tissue, the so-called normal-appearing white matter (NAWM). We performed differential gene expression analysis and quantitative RT-PCR on subcortical white matter from 11 multiple sclerosis and 8 control cases. Differentially expressed genes were further analysed in detail by in situ hybridization and immunofluorescence studies. We show that genes known to be involved in anti-inflammatory and protective mechanisms such as STAT6, JAK1, IL-4R, IL-10, Chromogranin C and Hif-1alpha are consistently upregulated in the multiple sclerosis NAWM. On the other hand, genes involved in pro-inflammatory mechanisms, such as STAT4, IL-1beta and MCSF, were also upregulated but less regularly. Immunofluorescence colocalization analysis revealed expression of STAT6, JAK1, IL-4R and IL-13R mainly in oligodendrocytes, whereas STAT4 expression was detected predominantly in microglia. In line with these data, in situ hybridization analysis showed an increased expression in multiple sclerosis NAWM of HIF-1alpha in oligodendrocytes and HLA-DRalpha in microglia cells. The consistency of the expression levels of STAT6, JAK1, JAK3 and IL-4R between the multiple sclerosis cases suggests an overall activation of the STAT6-signalling pathway in oligodendrocytes, whereas the expression of STAT4 and HLA-DRalpha indicates the activation of pro-inflammatory pathways in microglia. The upregulation of genes involved in anti-inflammatory mechanisms driven by oligodendrocytes may protect the CNS environment and thus limit lesion formation, whereas the activation of pro-inflammatory mechanisms in microglia may favour disease progression. Altogether, our data suggests an endogenous inflammatory reaction throughout the whole white matter of multiple sclerosis brain, in which oligodendrocytes actively participate. This reaction might further influence and to some extent facilitate lesion formation.

BACKGROUND

We aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.

METHODS

We selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel-Haenszel approach to account for heterogeneity between sample collections.

RESULTS

A previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 x 10-5), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.

CONCLUSIONS

Our results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.

OBJECTIVE

Genetic studies in the systemic sclerosis (SSc), an autoimmune disease that clinically manifests with dermal and internal organ fibrosis and small vessel vasculopathy, have identified multiple susceptibility genes including HLA-class II, PTPN22, IRF5, and STAT4 which have also been associated with other autoimmune diseases, such as systemic lupus erythematosus (SLE). These data suggest that there are common autoimmune disease susceptibility genes. The current report sought to determine if polymorphisms in the C8orf13-BLK region (chromosome 8p23.1-B lymphoid tyrosine kinase), which is associated with SLE, are associated also with SSc.

METHODS

Two variants in the C8orf13-BLK region (rs13277113 & rs2736340) were tested for association with 1050 SSc cases and 694 controls of North Americans of European descent and replicated in a second series 589 SSc cases and 722 controls from Spain.

RESULTS

The "T" allele at rs2736340 variant was associated with SSc in both the U.S. and Spanish case-control series (P = 6.8 x 10(-5), OR 1.27, 95% CI 1.1-1.4). The "A" allele at rs13277113 variant was associated with SSc in the U.S. series only (P = 3.6 x 10(-4), OR 1.32, 95% CI 1.1-1.6) and was significant in the combined analyses of the two series (P = 2.0 x 10(-3); OR 1.20, 95% CI 1.1-1.3). Both variants demonstrated an association with the anti-centromere antibody (P = 2.2 x 10(-6) and P = 5.5 x 10(-4), respectively) and limited SSc (P = 3.3 x 10(-5) and P = 2.9 x 10(-3), respectively) in the combined analysis. Peripheral blood gene expression profiles suggest that B-cell receptor and NFkappaB signaling are dysregulated based on the risk haplotype of these variants.

CONCLUSIONS

We identify and replicate the association of the C8orf13-BLK region as a novel susceptibility factor for SSc, placing it in the category of common autoimmune disease susceptibility genes.

Our adaptive immune system induces distinct responses to different pathogens because of the functional plasticity of dendritic cells (DCs); however, how DCs program unique responses remains unclear. Here, we found that the cytokine thymic stromal lymphopoietin (TSLP) potently transduced a unique T helper type 2 (T(H)2)-inducing compound signal in DCs. Whereas activation of nuclear factor kappaB (predominantly p50) drove DCs to produce OX40L to induce T(H)2 differentiation, the activation of signal transducer and activator of transcription 6 (STAT6) triggered DCs to secrete chemokines necessary for the recruitment of T(H)2 cells. In addition, TSLP signaling limited the activation of STAT4 and interferon regulatory factor 8 (IRF-8), which are essential factors for the production of the T(H)1-polarizing cytokine interleukin-12 (IL-12). By contrast, Toll-like receptor ligands and CD40 ligand did not activate STAT6 in myeloid DCs, but instead increased the abundance of STAT4 and IRF-8 to induce T(H)1 responses through the production of IL-12. Therefore, we propose that the functional plasticity of DCs relies on elaborate signal codes that are generated by different stimuli.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway is involved in immune function and cell growth. We evaluated the association between genetic variation in JAK1 (10 SNPs), JAK2 (9 SNPs), TYK2 (5 SNPs), suppressors of cytokine signaling (SOCS)1 (2 SNPs), SOCS2 (2 SNPs), STAT1 (16 SNPs), STAT2 (2 SNPs), STAT3 (6 SNPs), STAT4 (21 SNPs), STAT5A (2 SNPs), STAT5B (3 SNPs), STAT6 (4 SNPs) with risk of colorectal cancer. We used data from population-based case-control studies (colon cancer n = 1555 cases, 1,956 controls; rectal cancer n = 754 cases, 959 controls). JAK2, SOCS2, STAT1, STAT3, STAT5A, STAT5B, and STAT6 were associated with colon cancer; STAT3, STAT4, STAT6, and TYK2 were associated with rectal cancer. Given the biological role of the JAK/STAT-signaling pathway and cytokines, we evaluated interaction with IFNG, TNF, and IL6; numerous statistically significant associations after adjustment for multiple comparisons were observed. The following statistically significant interactions were observed: TYK2 with aspirin/NSAID use; STAT1, STAT4, and TYK2 with estrogen status; and JAK2, STAT2, STAT4, STAT5A, STAT5B, and STAT6 with smoking status and colon cancer risk; JAK2, STAT6, and TYK2 with aspirin/NSAID use; JAK1 with estrogen status; STAT2 with cigarette smoking and rectal cancer. JAK2, SOCS1, STAT3, STAT5, and TYK2 were associated with colon cancer survival (hazard rate ratio (HRR) of 3.3 95% CI 2.01,5.42 for high mutational load). JAK2, SOCS1, STAT1, STAT4, and TYK2 were associated with rectal cancer survival (HRR 2.80 95% CI 1.63,4.80). These data support the importance of the JAK/STAT-signaling pathway in colorectal cancer and suggest targets for intervention.

Single nucleotide polymorphisms in the STAT4 gene have recently been shown to be associated with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Primary Sjögren's syndrome (pSS) is a related autoimmune disease thought to have a pathogenesis similar to these diseases. To test the hypothesis that the variant haplotype of STAT4 seen in RA and SLE is also associated with pSS, we genotyped rs7574865, the most strongly disease-associated SNP in the variant STAT4 haplotype, in 124 Caucasian pSS subjects and compared them to 1143 Caucasian controls. The disease-associated T allele was more common in chromosomes of the pSS patients (29.6%) than in controls (22.3%), leading to a P-value for association of 0.01. These results implicate polymorphisms in the STAT4 gene in the pathogenesis of pSS.

We analyzed the molecular mechanisms by which IL-12 and IL-18 induce transcriptional activity of the IFN-gamma promoter in primary human CD4+ T cells. In transfection experiments, we found that IL-18 directly induces IFN-gamma promoter activity, whereas significant activation with IL-12 required costimulation with alphaCD3/CD28. Furthermore, IL-12 caused in vivo protection of a STAT4 (-236) binding site, whereas stimulation with IL-18 or IL-12 plus alphaCD3/CD28 induced occupancy of a downstream AP-1 site. Mutation of this AP-1 site abrogated both IL-12- and IL-18-mediated promoter activation, whereas mutation of the STAT site inhibited IL-12-dependent activation. These data suggest that both AP-1 and STAT4 are required for IL-12-dependent IFN-gamma promoter activity, whereas IL-18 causes direct activation via AP-1. This differential activation of the IFN-gamma promoter gives further insights into molecular pathways governing Th1 T cell development and differentiation.

Experimental allergic encephalomyelitis (EAE) is a CD4(+) Th1 cell-mediated inflammatory demyelinating autoimmune disease of the CNS that serves as an animal model for multiple sclerosis (MS). IL-12 is a proinflammatory cytokine that plays a crucial role in the induction of neural Ag-specific Th1 differentiation and pathogenesis of CNS demyelination in EAE and MS. Curcumin (1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a naturally occurring polyphenolic phytochemical isolated from the rhizome of the medicinal plant Curcuma longa. It has profound anti-inflammatory activity and been traditionally used to treat inflammatory disorders. In this study we have examined the effect and mechanism of action of curcumin on the pathogenesis of CNS demyelination in EAE. In vivo treatment of SJL/J mice with curcumin significantly reduced the duration and clinical severity of active immunization and adoptive transfer EAE. Curcumin inhibited EAE in association with a decrease in IL-12 production from macrophage/microglial cells and differentiation of neural Ag-specific Th1 cells. In vitro treatment of activated T cells with curcumin inhibited IL-12-induced tyrosine phosphorylation of Janus kinase 2, tyrosine kinase 2, and STAT3 and STAT4 transcription factors. The inhibition of Janus kinase-STAT pathway by curcumin resulted in a decrease in IL-12-induced T cell proliferation and Th1 differentiation. These findings highlight the fact that curcumin inhibits EAE by blocking IL-12 signaling in T cells and suggest its use in the treatment of MS and other Th1 cell-mediated inflammatory diseases.

Recent data support a modulatory role for CD4 T cells in experimental renal ischemia-reperfusion injury (IRI). CD4 T cells can functionally differentiate to either a Th1 (IFN-gamma producing) or the counterbalancing Th2 (IL-4) phenotype. The enzymes signal transducers and activators of transcription (STAT) 4 and STAT6 regulate Th1 or Th2 differentiation and cytokine production, respectively. We therefore hypothesized that mice that were STAT4 deficient would be protected from renal IRI and that STAT6-deficient mice would have a more severe course. Intracellular cytokine staining of splenocytes from STAT4-/- or STAT6-/- exhibited distinct IFN-gamma and IL-4 cytokine expression profiles. STAT6-/- had markedly worse renal function and tubular injury postischemia compared with wild type. STAT4-/- had only mildly improved function. Renal phagocyte infiltration and ICAM-1 upregulation were similar in STAT4-/-, STAT6-/-, and wild type. To evaluate if the mechanism of the marked worsening in the STAT6-/- mice could be due to IL-4 deficiency, IL-4-deficient mice were studied and had similar postischemic phenotype to STAT6-/- mice. These data demonstrate that the STAT6 pathway has a major protective role in renal IRI. IL-4 deficiency is a likely mechanism underlying the STAT6 effect. A "yin-yang" role for inflammation is emerging in renal IRI, similar to recent observations in atherosclerosis.

Berberine, an alkaloid derivative from Berberis vulgaris L., has been used extensively in traditional Chinese medicine to treat diarrhea and diabetes, but the underlying mechanisms for treating diabetes are not fully understood. Recent studies suggested that berberine has many beneficial biological effects, including anti-inflammation. Because type 1 diabetes is caused by T cell-mediated destruction of beta cells and severe islet inflammation, we hypothesized that berberine could ameliorate type 1 diabetes through its immune regulation properties. Here we reported that 2 weeks of oral administration of berberine prevented the progression of type 1 diabetes in half of the NOD mice and decreased Th17 and Th1 cytokine secretion. Berberine suppressed Th17 and Th1 differentiation by reducing the expression of lineage markers. We found that berberine inhibited Th17 differentiation by activating ERK1/2 and inhibited Th1 differentiation by inhibiting p38 MAPK and JNK activation. Berberine down-regulated the activity of STAT1 and STAT4 through the suppression of p38 MAPK and JNK activation, and it controlled the stability of STAT4 through the ubiquitin-proteasome pathway. Our findings indicate that berberine targets MAPK to suppress Th17 and Th1 differentiation in type 1 diabetic NOD mice. This study revealed a novel role of ERK in Th17 differentiation through down-regulation of STAT3 phosphorylation and RORgamma t expression.

TGF-beta1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-beta1's suppressive effects on CD4(+) T cell expression of IFN-gamma expression remain incompletely understood. To evaluate mechanisms by which TGF-beta1 inhibits IFN-gamma expression in CD4(+) T cells, we primed naive wild-type murine BALB/c CD4(+) T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-beta1. We found that the presence of TGF-beta1 during priming of CD4(+) T cells suppressed both IFN-gamma expression during priming as well as the development of Th1 effector cells expressing IFN-gamma at a recall stimulation. TGF-beta1 inhibited the development of IFN-gamma-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-beta1 can inhibit Th1 development by acting directly on the CD4(+) T cell. During priming, TGF-beta1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-gamma expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-beta1's inhibition of Th1 development, but did not prevent TGF-beta1's inhibition of IFN-gamma expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-beta1's inhibition of IFN-gamma expression during priming, but did not prevent TGF-beta1's inhibition of Th1 development. These data show that TGF-beta1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4(+) T cells at priming and at recall.

OBJECTIVE

To confirm and define the genetic association of STAT4 and systemic lupus erythematosus (SLE), investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5.

METHODS

30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in five new sets of cases and controls for replication. STAT4 cDNA was analysed by 5'-RACE PCR and sequencing. Expression levels were measured by quantitative PCR.

RESULTS

In the fine mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals, represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. Transcription of alternative tissue-specific exons 1, indicating the presence of tissue-specific promoters of potential importance in the expression of STAT4, was also detected. No interaction with associated SNPs of IRF5 was observed using regression analysis.

CONCLUSIONS

These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. The results also indicate that the genes STAT4 and IRF5 act additively to increase the risk for SLE.

OBJECTIVE

All-trans retinoic acid (ATRA), a potent derivative of vitamin A, can regulate immune responses. However, its role in inducing immune tolerance associated with the prevention of islet inflammation and inhibition of type 1 diabetes remains unclear.

METHODS

We investigated the mechanisms underlying the potential immunoregulatory effect of ATRA on type 1 diabetes using an adoptive transfer animal model of the disease.

RESULTS

Our data demonstrated that ATRA treatment inhibited diabetes in NOD mice with established insulitis. In addition, it suppressed interferon (IFN)-gamma-producing CD4(+) and CD8(+) T effector (Teff) cells and expanded T regulatory (Treg) cells in recipient mice transferred with diabetic NOD splenocytes, without affecting either interleukin (IL)-17--or IL-4-producing cells. Consistent with these results, ATRA reduced T-bet and STAT4 expression in T-cells and decreased islet-infiltrating CD8(+) T-cells, suppressing their activation and IFN-gamma/granzyme B expression. Depletion of CD4(+)CD25(+) Treg cells impaired the inhibitory effect of ATRA on islet-infiltrating T-cells and blocked its protective effect on diabetes. Therefore, ATRA treatment induced Treg cell-dependent immune tolerance by suppressing both CD4(+) and CD8(+) Teff cells while promoting Treg cell expansion.

CONCLUSIONS

These results demonstrate that ATRA treatment promoted in vivo expansion of Treg cells and induced Treg cell-dependent immune tolerance by suppressing IFN-gamma-producing T-cells, without affecting Th17 cells. Our study also provides novel insights into how ATRA induces immune tolerance in vivo via its effects on Teff and Treg cells.

Via cytoplasmic signal transduction pathways, cytokines induce a variety of biological responses and modulate the outcome of inflammatory diseases and malignancies. Crohn's disease is a chronic inflammatory bowel disease of unknown etiology. Perturbation of the intestinal cytokine homeostasis is believed to play a pivotal role, but the pathogenesis of Crohn's disease is not fully understood. Here, we study intestinal T cells from Crohn's disease and healthy volunteers. We show that STAT3 and STAT4 are constitutively activated in Crohn's patients but not in healthy volunteers. The activation is specific, because other STAT proteins are not constitutively activated. Furthermore, the STAT3 regulated protein, SOCS3, is also constitutively expressed in Crohn's patients but not in healthy volunteers. Taken together, these data provide evidence of abnormal STAT/SOCS signaling in Crohn's disease. This aberrant activation, so far noted only in malignant cells, establish a new critical approach for better understanding the immunopathogenesis of Crohn's disease.

IFN-gamma, a product of NK and T cells, is a key cytokine contributing innate and adaptive immunity. IFN-gamma production is induced via direct cell-cell contacts with APC and IFN-gamma -producing cells or by cytokines. During microbial infections macrophage-derived IFN-alpha, IL-12, and IL-18 enhance IFN-gamma production and Th1 response. Here we show that IFN-alpha in combination with IL-18 very efficiently induces IFN-gamma expression also in primary, nonactivated NK cells and in NK-92 cell line. Comparison of the kinetics of IFN-gamma mRNA expression in nonactivated NK cells, NK-92 cells and activated T cells stimulated with IFN-alpha or IL-12 revealed that, although both of these cytokines directly up-regulate IFN-gamma mRNA expression, its levels remain elevated much longer with IL-12 stimulation. In both NK cells and T cells, Stat4 is known to be critical in IL-12 and IFN-alpha signaling. We show that Stat4 activation is transient in cells stimulated with IFN-alpha, whereas IL-12 induces more long-lasting activation of the transcription factor. This prolonged activation of IFN-gamma gene by IL-12 may result in more efficient IFN-gamma production compared to that of IFN-alpha. Our results demonstrate that IFN-alpha and IL-18 are important innate cytokines in inducing NK cell IFN-gamma production.